1. Field of the Invention
The present invention relates generally to devices for performing electrophoresis and electro-blot transfer on gel slab assemblies. The present invention particularly relates to a device that can perform both vertical electrophoresis on protein and nucleic acid samples, and electro-blot transfer.
2. Related Art
Two of the most prominent techniques for separating macromolecules are chromatography and gel electrophoresis. Electrophoresis devices have evolved since the discovery that charged particles suspended between opposite poles and in an electric field migrate toward the pole possessing the charge opposite that particle.
Gel electrophoresis is primarily used to separate large macromolecules ranging in size from 1 to 10,000 KD (Kilodaltons). A kilodalton is a unit of molecular weight roughly equivalent to the mass of 1,000 hydrogen atoms. Gel electrophoresis can separate hundreds of macromolecules from one another while using less than a millionth of a gram of sample material.
Generally, the first step in conventional gel electrophoresis processes involves preparing a gel slab assembly. A gel slab is typically formed from an acrylamide solution. Typically, the acrylamide solution is cast between two glass plates separated by thin strips of plastic, (known as side spacers to those proficient in the arts). The cast acrylamide solution then polymerizes forming a pore matrix. Within the polymerized gel material, these pores form a "sieve" that retards the movement of macromolecules. Using a special comb, wells are formed at the uppermost part of the gel slab during casting. The areas below each well are termed "lanes."
Thereafter, a gel slab assembly is placed into an electrophoresis device. Samples are then placed in a narrow band within each well and an electric field is applied across the gel slab. Typically, the upper and lower portion of the gel assembly are submerged in separate isolated buffer solution reservoirs. A typical buffer solution is an SDS-Denaturing solution per Laemmli, U.K. (1970) Nature 277,680. The electric field induces the macromolecules of the samples to migrate through the gel slab. As the samples migrate, the macromolecules are "sieved" by molecular weight in their respective lanes. If the electrophoresis device is working properly, each species of macromolecules of a specific size will be in bands arranged from the top of the gel to the bottom, according to molecular weight, with the largest molecules at the top.
When performing "band" comparison between a plurality of samples, it is very important the adjacent bands represent similar macromolecules. One significant factor that controls the "band" representation from lane to lane is whether or not the electric field across the gel slab assembly is uniform. Two parameters that affect the consistency of the electric field are the current and temperature distribution across the gel slab. A non-uniform current and/or temperature distribution will cause the electric field to be stronger or weaker from one "lane" to another. As the electric field causes the macromolecules to migrate the macromolecules move down "lanes" with like macromolecules moving at different speeds if either the applied voltage or the temperature is nonuniform. These phenomena are commonly referred to as "smiles" or "frowns," with the smiling gel having the outside lanes moving slower than the central lanes, and a frown being the opposite, with the outside lanes moving faster creating the impression of a human "frown." Because visual band comparison must take place to obtain macromolecule size, and gels that are "smiling" or "frowning" are harder to compare. This is one significant problem with conventional electrophoresis devices.
As discussed earlier, the electric field is dependent on uniform current flow and uniform temperature distribution. These two parameters are intertwined. Generally, the higher the voltage applied to a given macromolecule, the faster the migration process takes place. Temperature, however, should not rise above about 60 degrees celsius, as degradation of some macromolecules will occur above this point. However, raising the voltage increases the heat generated in the gel slab. Therefore, a "trade-off" exists. Increased voltage yields desirable process times but undesirable higher gel slab temperatures and increased non-uniformity in temperature distribution.
Accordingly, with regard to current distribution, under ideal conditions, it is desirable to design a device such that the current draw is mainly through the gel slab, and not the surrounding buffer. Therefore, it is important that the positively charged and negatively charged buffer solutions be substantially isolated from each other. Conventional devices have complicated designs using rubber gaskets so as to ensure that the buffer solution reservoirs are isolated.
In addition, directing most of the current through the gel slab will decrease the overall energy dissipation. Decreased energy dissipation will reduce the overall gel slab temperature. This will ensure that the gel slab temperature is kept well below its degradation point.
With regard to temperature distribution, the problem in part, is a function of where the current is being dissipated. Therefore, a unit in which the current is non-uniform, the power will be dissipated non uniformly, causing "smiles" or "frowns." Additionally., under ideal conditions, with the current passing through the gel slab, there is a need to transfer heat from the gel to the surrounding buffer. Heat sinking the gel slab with the buffer reservoir will thereby maintain uniform temperature distribution.
Another significant problem with conventional electrophoresis devices resides in their ability to accept gel slabs of variable thickness in an unencumbered manner. This is because conventional devices are not flexible enough to easily accommodate a wide variety of gel assemblies as the complex assembly procedures, and number of parts used, hinders the user.
After electrophoresis with a non-prestained sample is performed, the samples, although present, are undetectable. Therefore, a detection technique must be utilized so that the samples can be analyzed.
One detection method involves staining and de-staining the gel slab. In this process, the entire gel slab is stained with a dye that only adheres to the macromolecules. Thereafter, a de-staining process is performed wherein dye not adhered to the macromolecules is washed away. The bands of macromolecules thus become visible.
Another common detection method involves the use of antibodies. In this method, the bands of proteins or samples are "transferred" or blotted to a macromolecule binding membrane. In order to detect the presence of certain proteins or samples on the membrane, a known antibody is introduced. The antibody will only combine with a specific protein or sample. In order to detect the antibody-protein combination, the antibodies are "tagged."
A common transfer process is called "electro-blot" transfer. In the "electro-blot" transfer process the macromolecules in the gel slab "migrate" under an electric field "on" to an electro-blot membrane. A commonly used membrane is nitrocellulose.
In designing an electro-blot transfer apparatus it is important that the blotting membrane be in close contact with the gel slab. Close contact between the gel slab and blotting membrane assures that a complete transfer of bands will occur with no significant loss of resolution. For example, the existence of air bubbles between the gel slab and blotting membrane will prevent the band images from being transferred. Additionally, like the electrophoresis devices, it is important to maintain an uniform electric field directed across the electro-blot sandwich.
In addition, preparation of the gel slab/nitrocellulose sandwich must be carefully performed so that the macromolecules on the gel membrane are not removed or contaminated. Therefore, the electro-blot apparatus must be designed so that the electro-blot sandwich can be easily assembled and disassembled.
Another problem with conventional electrophoresis and electro-blot transfer devices is that they can only perform either electrophoresis or electro-blot transfer. Having to purchase two separate units can be very costly. Furthermore, two devices can occupy more work space. This is a significant disadvantage with conventional devices.
The present invention was developed with certain objectives in mind. One objective was to develop an apparatus that could perform both electrophoresis and electro-blot transfer. Another objective was to develop an apparatus which could easily accommodate gel slab assemblies of variable thicknesses. A further objective was to develop an apparatus that would have a uniform electric field across the gel slab during electrophoresis and, using the same electrode placement, across the electro-blot sandwich during electro-blot transfer.
As will be shown below, the present invention meets the above objectives. Moreover, the present invention provides advantages heretofore unavailable in conventional devices.